It is proposed to undertake the systematic construction of a series of copper centers which reproduce the most important aspects of known copper chemistry in proteins, using techniques of de novo rational protein design. The first step is to construct faithful analogues of natural centers to study the factors that are required to introduce and control metal centers in a protein matrix. The second step is to systematically vary the metal geometry in the designs beyond that normally encountered in nature in order to explore structure/reactivity relationships. The construction of a new function in a protein requires that detailed local interactions are correctly predicted and formed, and that the overall, global fold and stability are maintained. The goal of this proposal is to focus specifically on the design of local interactions. The design a global fold is proposal is to focus specifically on the design of local interactions. The design a global fold is deliberately circumvented by using a known protein structure as a "scaffold" within which a completely new binding site is built by modifying its sequence substantially such that the new ligand binding site is accommodated without altering the overall backbone fold. A novel molecular modeling techniques applies this "inverse folding" approach by searching proteins of known-three dimensional structure for mutations that establish the primary metal coordination sphere and which resolve sterid conflicts between the new site and the surrounding protein matrix, while maintaining the original backbone fold. Designs are based on stereochemical definitions describing ranges of allowed geometrical relationships between residues and substrate. In view of their relative geometrical simplicity and small size, availability of several X-ray structures, extensive spectroscopic studies, and wealth of model work, important role in nature, copper sites present attractive targets for design studies using this new "site-grafting" approach. Furthermore, their electronic structure, and hence their reactivity, is controlled by the ability of the protein to present a rigid ligand sphere within which perturbed metal coordination geometries can be stably maintained. Static molecular modeling techniques are therefore particularly appropriate for their design. Spectroscopic studies of copper or other transition metals can be used to probe the nature of the coordination sphere in order to evaluate the success of the design and analyze the nature of the mistakes made in failures. Thioredoxin, a stable, monomeric E. coli protein of known structure has been chosen as the host protein into which the new centers will be constructed by site-directed mutagenesis. The construction of metal site, and systematic variation of coordination geometry within a protein matrix by de novo approach to the study of in metal centers in proteins. Ultimately this will lead to the construction of sites with reactivities not yet encountered in nature, and to the design of new catalysts for biotechnological applications.